pubs.acs.org/Langmuir © 2009 American Chemical Society
Competitive Protein Adsorption of Albumin and Immunoglobulin G from Human Serum onto Polymer Surfaces Maria Holmberg*,† and Xiaolin Hou‡ †
Department of Micro- and Nanotechnology and ‡Risø National Laboratory for Sustainable Energy, Technical University of Denmark, Frederiksborgvej 399, DK-4000 Roskilde, Denmark Received July 6, 2009. Revised Manuscript Received August 21, 2009
Competitive protein adsorption from human serum onto unmodified polyethylene terephthalate (PET) surfaces and plasma-polymerized PET surfaces, using the monomer diethylene glycol vinyl ether (DEGVE), has been investigated using radioactive labeling. Albumin and immunoglobulin G (IgG) labeled with two different iodine isotopes have been added to human serum solutions of different concentrations, and adsorption has been performed using adsorption times from approximately 5 s to 24 h. DEGVE surfaces showed indications of being nonfouling regarding albumin and IgG adsorption during competitive protein adsorption from diluted human serum solutions with relatively low protein concentrations, but the nonfouling character was weakened when less diluted human serum solutions with higher protein concentrations were used. The observed adsorption trend is independent of adsorption time, indicating that the protein concentration has a stronger influence on observed adsorption characteristics of the material than the adsorption time has.
Introduction A key aspect of many biomaterials is their nonfouling characteristics, or their ability to resist protein adsorption. Via prevention of this first step in the response from the human body upon introduction of an artificial material, the idea is that subsequent, often unwanted steps can be prevented, or at least delayed.1,2 The unwanted steps can lead to inflammation, thrombosis, and eventually rejection of the material by the human body. The nonfouling characteristics of a material are also connected to the blood compatibility of the material, where a blood compatible material can be described as a material that does not induce thrombosis when introduced into the body.3 Aside from nonfouling and blood compatibility, parameters such as elasticity and chemical and mechanical stability of the material are important when the use of a biomaterial in a biological system such as the human body is being evaluated. Often alteration or modification of a material is needed to produce a feasible biomaterial, and one of the most common and successful ways of surface modification for obtaining a nonfouling material is to attach polyethylene oxide (PEO) molecules to the surface of the material, a technique also called PEG (polyethylene glycol) grafting.4-7 The resistance to protein adsorption observed on these modified surfaces is most often attributed to steric repulsion effects, based on the increased *To whom correspondence should be addressed: Department of Microand Nanotechnology, Technical University of Denmark, Frederiksborgvej 399, Building 124, DK-4000 Roskilde, Denmark. Telephone: þ45 4677 4735. E-mail:
[email protected]. (1) Anderson, J. M.; Rodriguez, A.; Chang, D. T. Semin. Immunol. 2006, 20, 86– 100. (2) Gray, J. J. Curr. Opin. Struct. Biol. 2004, 14, 110–115. (3) Ratner, B. D.; Hoffman, A. S.; Schoen, F. J.; Lemons, J. E. Biomaterial Science. An introduction to Materials in Medicine, 2nd ed.; Elsevier Academic Press: San Diego, 2004. (4) Jeon, S. I.; Lee, J. E.; Andrade, J. D.; de Gennes, P. G. J. Colloid Interface Sci. 1991, 142(1), 149–158. (5) Morra, M.; Occhiello, E.; Garbassi, F. Clin. Mater. 1993, 14, 255–265. (6) Fukai, R.; Dakwa, P. H. R.; Chen, W. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 5389–5400. (7) Hansson, K. M.; Tosatti, S.; Isaksson, J.; Wetter€o, J.; Textor, M.; Lindahl, T. L.; Tengvall, P. Biomaterials 2005, 26, 26861–26872.
938 DOI: 10.1021/la902409n
hydrophilicity of the surface and flexibility of the grafted PEO chains.8-12 Both chemical and physical techniques are used to modify surfaces with PEO molecules, and while the approach of performing physical modification often is relatively easy and can be performed in one step, there is often an inherited instability of the modified layer caused by the lack of covalent linkage between the sample surface and PEO molecules.13 Chemical modification gives strong covalent bonds between surface and PEO molecules but is often tedious to perform, and the modification can be difficult to execute in routine.5,14 So-called “soft” plasma polymerization has been suggested as an alternative technique for modifying surfaces for being nonfouling.15-18 By using monomers with oligo(ethylene glycol) groups, a PEO-like layer can be formed on the sample surface, and these PEO-like layers have been shown to reduce the level of adsorption of protein onto the surfaces.10,19-22 In this study, we have performed soft plasma (8) Muir, B. W.; Tarasova, A.; Gengenbach, T. R.; Menzies, D. J.; Meagher, L.; Rovere, F.; Fairbrother, A.; McLean, K. M.; Hartley, P. G. Langmuir 2008, 24, 3828–3835. (9) Szleifer, I. Curr. Opin. Solid State Mater. Sci. 1997, 2, 337–344. (10) Chu, L. Q.; Knoll, W.; F€orch, R. Chem. Mater. 2006, 18, 4840–4844. (11) Zhang, Z.; Zhang, M.; Chen, S.; Horbett, T. A.; Ratner, B. D.; Jiang, S. Biomaterials 2008, 29, 4285–4291. (12) Unsworth, L. D.; Sheardown, H.; Brash, J. L. Langmuir 2008, 24, 1924– 1929. (13) Nylander, T.; Samdshina, Y.; Lindman, B. Adv. Colloid Interface Sci. 2006, 123-126, 105–123. (14) Kingshott, P.; Wei, J.; Bagge-Ravn, D.; Gadegaard, N.; Gram, L. Langmuir 2003, 19, 6912–6921. (15) Kumar, D. S.; Fujioka, M.; Asano, K.; Shoji, A.; Jayakrishnan, A.; Yoshida, Y. J. Mater. Sci.: Mater. Med. 2007, 18, 1831–1835. (16) Bouaidat, S.; Berendsen, C.; Thomsen, P.; Petersen, S. G.; Wolff, A.; Jonsmann, J. Lab Chip 2004, 2, 632–637. (17) Johnston, E. E.; Bryers, J. D.; Ratner, B. D. Langmuir 2005, 21, 870–881. (18) Siow, K. S.; Britcher, L.; Kumar, S.; Griesser, H. J. Plasma Processes Polym. 2006, 3, 392–418. (19) Holmberg, M.; Stibius, K. B.; Larsen, N. B.; Hou, X. J. Mater. Sci.: Mater. Med. 2008, 19, 2179–2185. (20) Wu, Y. J.; Timmons, R. B.; Jen, J. S.; Molock, F. E. Colloids Surf., B 2000, 19, 235–248. (21) Bremmell, K. E.; Kingshott, P.; Ademovic, Z.; Winther-Jensen, B.; Griesser, H. J. Langmuir 2006, 22, 313–318. (22) Bretagnol, F.; Lejeune, M.; Papadopoulou-Bouraoui, A.; Hasiwa, M.; Rauscher, H.; Ceccone, G.; Colpo, P.; Rossi, F. Acta Biomater. 2006, 2, 165–172.
Published on Web 09/04/2009
Langmuir 2010, 26(2), 938–942
Holmberg and Hou
polymerization with the monomer DEGVE to fabricate hydrophilic polymer surfaces with a high content of oligo(ethylene glycol) species that should show nonfouling character. Surface modification of a biomaterial is required for the material to be stable and functional in biological fluids, for example, in blood, which has an extremely complex character. Blood contains a large number of different proteins, some of them present at rather high concentrations, and each protein can interact differently with a specific surface. However, investigating the general resistance to protein adsorption can be a valuable and informative starting point for evaluating a possible biomaterial. Here we present results from competitive adsorption of protein onto polymer surfaces from human serum, where adsorption of albumin and immunoglobulin G (IgG) onto the surfaces is monitored simultaneously using radioactive labeling with two different iodine isotopes.19,23-25
Materials and Methods Polyethylene terephthalate (PET) film (Trafoma A/S, Denmark) was cut into disks with a diameter of 13 mm and used as the substrate. PET disks were modified using soft AC (alternating current) plasma polymerization26 with the monomer diethylene glycol vinyl ether (DEGVE). The PET disks were placed into the plasma polymerization chamber and pretreated with argon gas. Thereafter, the DEGVE monomer was introduced into the chamber and an alternating current was applied to the system. The polymerization was performed using a power of approximately 1.4 W for 30 min, resulting in a surface more hydrophilic than the bare PET surface. The thickness of the polymerized layer is estimated to be ∼100-200 nm. Both unmodified and modified surfaces were routinely characterized using an OCA 15 plus contact angle microscope (Dataphysics Instruments GmbH), a Perker-Elmer Spectrum One Fourier-transform infrared (FTIR) spectrometer (PerkinElmer Instruments), and a monochromatic X-ray photoelectron spectrometer (XPS) (K-Alpha from Thermo Fisher Scientific) before radioactive labeling experiments. More details on characterization of the unmodified and modified surfaces has been reported elsewhere.19 The roughness and surface topography of PET and DEGVE surfaces were analyzed using atomic force microscopy (AFM) (MultiMode Nanoscope III AFM, Vecco Instruments) in tapping mode in air. Roughness data were obtained from a surface area of 1 μm 4 μm, and the software package SPIP (Image Metrology A/S) was used for analysis of obtained AFM data, where linewise correction (LMS fit) of the first order was done before the roughness (root-mean-square roughness, Sq) of the surface was obtained. Albumin, IgG, human serum, and phosphate-buffered saline (PBS) were obtained from Sigma-Aldrich. Human serum diluted from approximately 90 to 0.1% in PBS buffer with added [125I]albumin and [131I]IgG was used for adsorption experiments. The amount of unlabeled albumin and IgG was at least 1000 times higher than the amount of labeled albumin and IgG in the different human serum solution used during adsorption. Albumin and IgG were labeled using the Iodo-Gen method, in which 0.15 mL of protein solution and 0.05 mL of iodine solution (125I or 131I, Perkin-Elmer Life and Analytical Science) were added to an Iodo-Gen tube (Pierce) for iodine labeling in room temperature for 15 min. The labeled proteins were separated from free iodine and other chemical reagents using gel chromatography (a PD-10 desalting column from Amersham Bioscience), and labeled proteins were checked for radiolysis using TCA (23) Nonckreman, C. J.; Rouxhet, P. G.; Dupont-Gillain, C. C. J. Biomed. Res. 2007, 81A, 791–802. (24) Brash, J. L.; Samak, Q. M. J. Colloid Interface Sci. 1978, 65(3), 495–504. (25) L^e, M. T.; Dejardin, P. Langmuir 1998, 14, 3356–3364. (26) Ademovic, Z.; Wei, J.; Winther-Jensen, B.; Hou, X.; Kingshott, P. Plasma Processes Polym. 2005, 2, 53–63.
Langmuir 2010, 26(2), 938–942
Article (trichloroacetic acid) precipitation.27 The amount of detected free iodine (125I and 131I) in human serum solutions used for adsorption was less than 5% (for each iodine isotope) during the experiments. Adsorption was performed at room temperature (∼20 °C); dry polymer substrates were placed in ELISA wells, to which 0.5 mL of human serum solution was added for adsorption. The disks were placed flat on the bottom of the wells when the solution was introduced. Control experiments in which one side of the disks has been covered with blue tape, thereby preventing adsorption, have shown that there is no significant difference in the amount of protein adsorbed between the side turning upward and the side turning downward. Thus, the area of both sides of the disks was used for calculating the amount of protein adsorbed per unit area. When adsorption had been carried out for the adsorption time intended, the protein solution was removed from the ELISA well with a pipet. Immediately after the removal of the protein solution, 2 mL of PBS buffer was added to the well, and a preliminary rinse of the disks was preformed by moving the ELISA plate from side to side before the added buffer was replaced with fresh buffer. The rinsing procedure in the ELISA wells was repeated three times before the disks were removed from the ELISA wells one by one and immersed in three beakers of fresh PBS buffer and rinsed, in one beaker at a time, before being placed in a plastic vial for radioactivity counting. The carryover of bulk solution on the rinsed surfaces was checked by counting the radioactivity in the solution from the last beaker used during rinsing. The radioactivity of 125I and 131I on each surface was measured with a Canberra 20 gamma counter (Canberra). By counting activities (Ast) of 125I and 131I in the standard solutions (protein solutions used for adsorption) with the calculated amount of protein (mst) and the activity of the sample disk (As), we obtained the amount of protein on the sample surface (ms) with the following formula: ms ¼ ðmst =Ast ÞAs With the known surface area of the sample disk (Ss), the protein adsorbed per unit was obtained as ms/Ss. It should be kept in mind that the exact concentration of albumin and IgG in the human serum dilutions is unknown, but via dilution of human serum that originates from the same lot (obtained from Sigma-Aldrich) for all concentrations used in the experimental setup, the relative percentage concentration between the different human serum solutions can be considered valid. When calculating the expected albumin and IgG concentration in different human serum dilutions, we used concentrations of 40 mg/mL albumin and 12 mg/ mL IgG for 100% human serum. The contribution from labeled protein was based on protein solutions of known concentrations, where the extent of loss during labeling and purification is estimated to be 20% for albumin and IgG. The 125I activity was measured by counting the total of its γ rays and X-rays in the low energy range (27-36 keV), and the 131I activity was measured by counting its γ rays at 364 keV. However, the X-rays of 131I (29-34 keV) as well as its Compton background and Bremsstrahlung continuum in the energy range of 27-36 keV interfere with the determination of the 125I activity when the two isotopes coexist in a sample. Therefore, by measuring the count rate of the 354 keV γ rays, as well as the count rate in the energy range of 27-36 keV of a pure 131I standard, we were able to retract the contribution from the 131I isotope from the 125I activity. Since the activity of 131I used in the experiments normally is lower than that of 125I and the counting efficiency for the energy range of 26-37 keV for 131I (
